Aspects of embryo-maternal communication in establishment of pregnancy in cattle
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DOI: 10.21451/1984-3143-AR2019-0075
Proceedings of the 33rd Annual Meeting of the Brazilian Embryo Technology Society (SBTE); Ilha de Comandatuba,
BA, Brazil, August 15th to 19th, 2019.
Aspects of embryo-maternal communication in establishment of pregnancy in cattle
José M. Sánchez, Constantine A. Simintiras, Patrick Lonergan*
School of Agriculture and Food Science, University College Dublin, Belfield, Dublin 4, Ireland.
Abstract established in commercial embryo transfer following the
transfer of 6-7 day old embryos, often produced in vitro,
Establishment of pregnancy in mammals to synchronized recipients. Indeed, pregnancy can be
requires reciprocal molecular communication between established following the transfer of elongated
the conceptus and endometrium that modifies the conceptuses up to at least Day 16 (Betteridge et al.,
endometrial transcriptome and uterine luminal milieu to 1980; Kimura and Matsuyama 2014) indicating that the
support pregnancy. Due to the small size of the early uterus does not need to receive any signals from the
embryo and elongating conceptus relative to the volume embryo prior to that stage in order for pregnancy occur.
of the uterine lumen, collection of endometrium adjacent Up to the blastocyst stage (Day 7-8), the
to the developing conceptus is difficult following bovine embryo is relatively autonomous, as blastocysts
conventional uterine flushing methods in cattle. Use of can be produced in vitro in the absence of contact with
endometrial explants in culture can overcome this the female reproductive tract and are capable of
challenge and reveal information about the dialogue establishing pregnancy after transfer to a synchronous
between the developing embryo and the uterus. The aim uterus. In contrast to primates and rodents, in which
of this short review is to summarize some of our recent implantation occurs shortly after the blastocyst enters
findings in relation to embryo maternal interaction during the uterus, in ungulates, such as ruminants and pigs, the
bovine pregnancy establishment and to put them in the early conceptus undergoes a phase of rapid growth and
wider context of fertility in cattle. elongation before implantation, the latter occurring 2-3
weeks after fertilization. In cattle, conceptus elongation is
Keywords: conceptus, fertility, bovine, uterus, initiated around Day 13 of gestation when the hatched
progesterone. bovine blastocyst transitions sequentially from a
spherical- to ovoid-, then tubular- and finally
Introduction filamentous-shaped structure that primarily involves
proliferation of the conceptus trophectoderm cells.
Embryo mortality is a major contributor to During this time, the elongating conceptus secretes
poor reproductive efficiency in dairy and beef cows. A interferon tau (IFNT), the maternal pregnancy
significant proportion of embryonic loss in cattle, recognition signal in ruminants (Bazer and Thatcher
2017). While there is a strong positive correlation
particularly lactating dairy cows, occurs during the first
between conceptus length and IFNT secretion (Rizos et
2-3 weeks after conception, before maternal recognition
al., 2012), surprisingly, threshold concentrations of IFNT
of pregnancy, which occurs around Day 16. In a recent
required to overcome luteolysis are as yet not known
comprehensive review, Wiltbank et al. (2016) described
(Forde and Lonergan 2017; Spencer et al., 2017).
four pivotal periods of pregnancy loss during the first
Elongation is necessary to ensure sufficient
trimester of gestation and discussed possible causes for
concentrations of IFNT are secreted and to expand the
pregnancy failure during these periods. Despite a
conceptus surface area for maximal vascular exchange
relatively high fertilization rate (>85%), 20%-50% of
with maternal tissues after implantation. An inability of
high-producing lactating dairy cows experience the conceptus to optimally elongate undoubtedly results
pregnancy loss during the first week of gestation. From in embryonic loss and is believed to significantly
Days 8 to 27, concomitant with embryo elongation and contribute to reproductive failure in cattle (Wiltbank et
maternal recognition of pregnancy, losses average al., 2016; Moraes et al., 2018). In contrast to pre-
approximately 30%. From Days 28 to 60, losses of hatching development, elongation is predominantly
approximately 12% occur while in the fourth period, maternally-driven, dependent on substances in the
during the third month of pregnancy, pregnancy losses uterine lumen fluid (ULF; or histotroph). As evidence
are reduced (approximately 2%), but may be elevated in for this, blastocysts do not elongate in vitro (Brandão et
some cows, particularly in those carrying twins in the al., 2004) and the absence of uterine glands in vivo
same uterine horn (Wiltbank et al., 2016). results in failure of blastocysts to elongate following
Communication between the developing embryo transfer (Gray et al., 2002).
embryo and the mother is vital for the successful Spatial and temporal changes of the
establishment and maintenance of pregnancy but the endometrial transcriptome and histotroph composition
requirement for this dialogue is temporal in nature and are necessary to establish uterine receptivity to
in reality only becomes absolutely essential from around implantation and, in turn, are pivotal to the likelihood of
Day 15-16 onwards. Pregnancies are routinely successful pregnancy in cattle. Those modifications are
_________________________________________
*Corresponding author: pat.lonergan@ucd.ie
orcid.org/0000-0001-5598-5044
Received: June 5, 2019 Copyright © The Author(s). Published by CBRA.
Accepted: June 27, 2019 This is an Open Access article under the Creative
Commons Attribution License (CC BY 4.0 license)Sánchez et al. Pregnancy establishment in cattle.
primarily regulated by the steroid hormone progesterone The capacity of the uterus to stimulate
(P4) derived from the corpus luteum (CL) which acts conceptus elongation is primarily dependent on
via the endometrium to promote conceptus growth and secretions from the luminal and glandular epithelium.
implantation, as well as conceptus-derived IFNT, which However, the timing of conceptus elongation is clearly
prevents development of the endometrial luteolytic associated with concentrations of P4 in circulation,
mechanism (Forde and Lonergan 2012; Brooks et al., which acts via the uterus (Clemente et al., 2009) to alter
2014). The role of P4 in uterine receptivity is the timing of PGR downregulation and thus onset of
unequivocal (Lonergan et al., 2016; Spencer et al., expression of key genes required for elongation in cattle
2016). Low circulating P4 concentrations in the first (Forde et al., 2009) and sheep (Satterfield et al., 2006).
week after ovulation, as frequently occurs in high- Consequently, P4 has an indirect effect on the secretion
producing lactating dairy cows, are associated with of IFNT by the conceptus, given the strong positive
under-developed conceptuses (Forde et al., 2012) with correlation between conceptus length and IFNT
an altered transcriptomic signature (Barnwell et al., production (Rizos et al., 2012). In order for P4 output
2016) and a low likelihood of establishing pregnancy from the CL to be maintained, sufficient quantities of
(Wiltbank et al., 2016). On the other hand, elevated IFNT must be produced by the conceptus by Day 16 to
concentrations of circulating P4 in the period abrogate the luteolytic mechanism and maintain CL
immediately after conception have been associated with function and induce expression of both classical and
advanced conceptus elongation (Carter et al., 2008), nonclassical interferon-stimulated genes (ISG) in the
increased IFNT production (Mann and Lamming 2001), different cellular compartments of the endometrium that
and greater pregnancy rates in cattle and sheep are proposed to regulate conceptus elongation.
(Ashworth et al., 1989; Stronge et al., 2005; McNeill et Temporal changes in uterine gene expression
al., 2006). Despite the definitive association between P4 occur irrespective of whether the cow is pregnant or not
and conceptus elongation, significant natural variation (Forde et al., 2009) and it is only during maternal
in age-matched in vivo- (Betteridge et al., 1980) and in recognition of pregnancy, around Day 16, by which
vitro-derived conceptuses occurs, even amongst time the conceptus is secreting copious amounts of
conceptuses developing in the same uterus (Clemente et IFNT (Forde and Lonergan 2017) that major changes in
al., 2009; Sánchez et al., 2019a; b). This would suggest gene expression between cyclic and pregnant
that part of the ability to elongate is intrinsic to the endometrium become apparent (Forde et al., 2011;
embryo and may be related to oocyte and/or blastocyst Bauersachs et al., 2012).
quality.
The aim of this short review is to summarize Blastocyst-induced changes in the endometrium
some of our recent findings in relation to embryo
maternal interaction during bovine pregnancy Pregnancy recognition in cattle is initiated
establishment and to put them in the wider context of around Day 15-16, both at the physiological and
fertility in cattle. Several of the recent studies referred to transcriptomic level. Nonetheless, the first week of
below have used an endometrial explant co-culture development is critical as evidenced by the fact that, at
system to elucidate this fine dialogue by examining least in high-producing dairy cows, about 50% of
changes in endometrial gene expression induced by embryos are no longer viable by Day 6-7 (Sartori et al.,
blastocysts (Passaro et al., 2018, 2019) or by an 2010). Whether communication between the embryo
elongating conceptus (Mathew et al., 2019; Sánchez et and endometrium at this stage is really important
al., 2019b; Bagés-Arnal et al., 2019). Due to the remains to be demonstrated convincingly. There is
maintenance of normal cellular and extracellular unequivocal evidence that when development occurs in
architecture in endometrial explants (Borges et al., vivo, blastocyst quality is improved in terms of
2012), some of the limitations of traditional cell culture ultrastructure (Rizos et al., 2002a), gene expression
can be overcome; for example, uterine explants allow profiles (Lonergan et al., 2003a, b; Gad et al., 2012),
the communication between resident populations of cryotolerance (Rizos et al., 2002b) and pregnancy rate
endometrial cells which cannot be achieved with current after transfer (Hasler et al., 1995) compared to when
2D and 3D cell culture technologies. blastocysts are produced in vitro. However, evidence of
a reciprocal effect of a single embryo on the cells of the
Role of progesterone in uterine receptivity and uterus is more difficult to detect. As mentioned earlier,
conceptus elongation the fact that blastocysts can be produced routinely in
vitro in the absence of contact with the reproductive
Progesterone from the CL induces both tract and subsequently establish a pregnancy after
temporal and spatial changes in the endometrial transfer to a recipient supports the notion that exposure
transcriptome necessary to establish uterine receptivity, of the reproductive tract to the early embryo, or vice-
when implantation in the uterus is possible (Forde et al., versa, is not required for pregnancy.
2009). These changes include down-regulation of the In vitro studies have demonstrated that
nuclear progesterone receptor (PGR) in the luminal and preimplantation embryos secrete a variety of
then glandular epithelium (Okumu et al., 2010), which biochemical messengers, embryotropins, that act in an
allows expression of genes and secretion of their protein autocrine manner to promote embryonic development
products, as well as active transport of other molecules, (reviewed by Wydooghe et al., 2015). For many of
required for conceptus elongation. these factors, expression of corresponding receptors in
Anim. Reprod., v.16, n.3, p.376-385, Jul./Sept. 2019 377Sánchez et al. Pregnancy establishment in cattle.
the uterus has been identified, the activation of which 2019b; Fig. 1) indicated that all of the DEG induced in
could lead to cellular and tissue responses in regions the endometrium by blastocyst-stage embryos are
that are in close physical contact with the embryo. IFNT-stimulated, in contrast to Day 15 when a
Others have reported that the early bovine embryo (from significant number of IFNT-independent genes are
Day 5 to Day 9) induces an anti-inflammatory response induced (Mathew et al., 2019; Sánchez et al., 2019b –
in uterine epithelial cells and immune cells in vitro see below).
(Talukder et al., 2017). Therefore, if factors secreted by These results support the concept that the early
the pre-elongating embryo enhance changes in the embryo is capable of communicating with the
transcriptome and in the proteome of the endometrium, reproductive tract. The effect on the endometrial
those changes are most likely to be local in nature and transcriptome is dependent on the stage of embryo
may not be detectable using crude methods of sample development and appears to be due solely to IFNT. The
collection. Use of an explant model allows the functional significance, if any, of such induced changes
interrogation of cells that were in direct contact with the remains to be fully elucidated given that it is possible to
embryo(s) facilitating the detection of such local transfer embryos from Day 7 onwards to a uterus that
embryo-induced changes in the endometrium during the has not previously been exposed to an embryo and
very early stages of pregnancy. achieve normal pregnancy rates.
Recently, local embryo-induced alterations in
the endometrial transcriptome from spatially-defined Response of the endometrium to in vivo or in vitro
regions in response to the presence of a Day 7 bovine derived conceptuses
embryo were reported (Sponchiado et al., 2017). In that
study, the presence of an embryo altered the abundance It has been elegantly shown that the
of 12 transcripts in the cranial part of the uterine horn endometrium can act as a ‘sensor’, with its
ipsilateral to the CL, including classical ISG (ISG15, transcriptome reflective of the type and developmental
MX1, MX2, OAS1Y), genes involved in prostaglandin competency of the conceptus present (Bauersachs et al.,
biosynthesis (PTGES, HPGD, AKR1L4), water channels 2009; Mansouri-Attia et al., 2009). These studies
(AQP4) and a solute transporter (SLC1A4); however, compared the endometrial responses to bovine
the extent of change was relatively minor in nature conceptuses produced by somatic cell nuclear transfer,
ranging from 1.35- to 2-fold). Based on this, we in vitro fertilization or artificial insemination (AI) and
hypothesized that the blastocyst induces local changes suggested that placental failure in bovine clone
in the endometrial transcriptome through the production pregnancies may originate from abnormal embryo-
of IFNT and potentially other diffusible factors. Using maternal communication during the peri-implantation
co-culture of endometrial explants in the absence or period (Day 18-20).
presence of blastocysts or medium conditioned by As stated above, it is generally accepted that
blastocysts, we demonstrated that bovine endometrium blastocysts produced in vitro are inferior in quality to in
responds to the presence of 8-day old blastocysts by vivo-derived embryos. This difference is reflected in the
upregulating expression of classical ISG (Passaro et al., fact that in commercial embryo transfer, the majority of
2018). This effect was (i) specific to the blastocyst stage in vitro-produced blastocysts are transferred fresh while
- earlier stages did not induce gene expression changes, the majority of in vivo-derived blastocysts are
(ii) dependent on the number of blastocysts present - a transferred frozen (Viana, 2018).
minimum of 5 blastocysts were required to detect such Mathew et al. (2019) compared the
changes, and (iii) independent of direct contact - the transcriptomic response of the endometrium following
effect was induced by embryos co-cultured on exposure to IFNT or a conceptus derived from the
endometrial explants using a cell culture insert transfer of an in vivo-derived (superovulation and AI) or
(preventing direct contact) as well as by blastocyst- in vitro-produced (IVF) blastocyst in order to identify
conditioned medium (Passaro et al., 2018). While others novel transcripts dependent and independent on IFNT,
have reported differential expression of a small number conceptus origin and conceptus sex. IVF- or AI-
of other transcripts in the endometrium in vivo, induced produced blastocysts were transferred into recipient
by the presence of a single blastocyst (Sponchiado et heifers on Day 7 of the estrous cycle. On Day 15, IVF-
al., 2017), or in cultured endometrial cells (Talukder et or AI-derived conceptuses were obtained by uterine
al., 2017; Gómez et al., 2018), we failed to detect in flushing and individually placed on endometrial
endometrial explants using qPCR (Passaro et al., 2018). explants in media for 6 h. Explants were also cultured
To extend these findings, Passaro et al. (2019) with media alone as a control or media containing 100
used RNA sequencing to investigate global changes in ng/mL recombinant ovine IFNT. Incubation of
the transcriptome of endometrial explants induced by endometrium with IFNT or IVF- or AI-derived
exposure to blastocysts. Exposure of bovine conceptuses altered the expression of 491, 498 and 576
endometrium to blastocyst-stage embryos resulted in the transcripts, respectively, compared to the control.
upregulation of 40 transcripts in blastocyst-exposed Further, 369 DEG were common between explants
endometrial explants compared to the control. exposed to IFNT or a conceptus. 240 DEG were
Comparison of this list of differentially expressed genes uniquely altered by conceptuses (IVF- and AI-derived)
(DEG) with the common list of genes altered in but not IFNT. Of these transcripts, 46 were shared
endometrial explants following culture with 100 ng/ml between the IVF and AI groups, while 61 and 133 were
IFNT or a Day 15 conceptus (from Sánchez et al., specific to IVF and AI conceptuses, respectively. Five
378 Anim. Reprod., v.16, n.3, p.376-385, Jul./Sept. 2019Sánchez et al. Pregnancy establishment in cattle.
genes (MLPH, PROM2, MYADM, VN1R4L, HTR1A) conceptus transcriptome mentioned above describing
were more abundant in endometrium exposed to female differential patterns of mRNA expression between short
compared to male conceptuses while a single gene (mean length of 4.2 ± 0.1mm) and long (24.7 ± 1.9 mm)
(ARL4C) was more abundant in response to male bovine conceptuses recovered on Day 15 of gestation
conceptuses than female conceptuses. (Barnwell et al., 2016). In that study, a total of 348
These data support the hypothesis that genes were differentially expressed related to
conceptus regulation of gene expression in the metabolism and biosynthesis. These genes and cellular
endometrium is complex and involves factors other than pathways involved in enhanced conceptus elongation, as
IFNT that may have a biological role in pregnancy well as the endometrial response blueprint to short and
establishment. The findings are consistent with the long conceptuses (Sánchez et al., 2019b), may
presence of unique proteins in ULF of pregnant heifers ultimately serve as markers of successful pregnancy.
on Day 16 and produced by short-term in vitro cultured Whether or not smaller conceptuses on a given
Day 16 conceptuses (Forde et al., 2015) and those of day are actually abnormal or whether they are simply
Bartol et al. (1985) who demonstrated that the fully slower in development is unclear; however, it is likely
elongated bovine conceptus produces a significant that they are compromised compared to longer (normal)
number of proteins when cultured in vitro. Further, conceptuses. Definitive proof will come from the
Spencer et al. (2013) demonstrated that the bovine recovery and retransfer of long and short age-matched
conceptus produces prostaglandins, which can modify conceptuses to establish their ability to initiate and
the endometrium prior to pregnancy recognition. maintain pregnancy.
While the differences in conceptus length are
Effect of conceptus length on the endometrial due, at least in part, to intrinsic differences in the
response embryo/conceptus, likely related to oocyte quality, it
would be wrong to completely discount a role for the
Significant variation in the length and uterus in contributing to variation in conceptus length
morphology of age-matched conceptuses exists, even and pregnancy establishment. Using a model of repeated
when multiple conceptuses are recovered from the same embryo transfer originally described by McMillan et al.
uterine environment (Clemente et al., 2009; O’Hara et (1999), Geary et al. (2016) classified heifers based on
al., 2014), despite the fact that embryos were produced pregnancy success following serial embryo transfer as
in vitro under the same conditions until the blastocyst high fertile (HF), subfertile (SF), or infertile (IF).
stage and were of similar morphological quality at the Conceptus survival and growth to Day 14 was not
time of transfer on Day 7. Conceptus length on a given compromised in SF and IF heifers. However, pregnancy
day in the period around pregnancy recognition is rate on Day 28 was higher in HF (70.4%) than in heifers
thought to be indicative of its quality and the likelihood with low fertility (36.8%; SF and IF). In a follow-up
of establishing and maintaining a pregnancy (Barnwell study (Moraes et al., 2018), pregnancy rate on Day 17
et al., 2016), although this has yet to be definitively was substantially higher in HF (71%) and SF (90%)
established. While significant differences in the than IF (20%) heifers. Furthermore, elongating
transcriptomes of long and short Day 15 conceptuses conceptuses were about twofold longer in HF than SF
have been reported (Barnwell et al., 2016), the heifers. Taken together, these data suggest that the
interaction between such divergent conceptuses and the uterus impacts conceptus survival and programs
endometrium had, until recently, not been described. conceptus development, and effects of dysregulated
We hypothesized that bovine endometrium exposed to conceptus-endometrial interactions elicit loss of the
long vs. short Day 15 conceptuses would exhibit a post-elongation conceptus in SF cattle during the
different transcriptome profile reflective of potential for implantation period of pregnancy.
successful pregnancy establishment. To test this In summary, bovine endometrium responds
hypothesis we used a combination of in vitro production differently in terms of its gene expression signature to
of bovine blastocysts, multiple embryo transfer and age-matched long and short conceptuses, in an IFNT-
conceptus-endometrial explant co-culture to investigate dependent and independent manner, which may be
the response of the endometrium to age-matched critical for embryo survival. In particular, short
conceptuses of different sizes collected from the same conceptuses failed to alter the expression of a large
uterine environment (Sánchez et al., 2019b). The main number of ISG that were altered by both IFNT and long
findings were that: (i) Day 15 conceptuses vary conceptuses, suggesting that insufficient IFNT
significantly in length, even when derived from the production is a major contributory factor to lower
same uterine environment; and (ii) the endometrium survival of such conceptuses. Furthermore, the
responds in an IFNT-dependent and independent alteration of >100 endometrial transcripts uniquely by
manner to conceptuses of different sizes which likely long conceptuses suggests that other aspects of
reflects the ability to successfully establish pregnancy maternal-embryo communication at this critical time are
(Fig. 1). These data complement nicely the data on the IFNT-independent.
Anim. Reprod., v.16, n.3, p.376-385, Jul./Sept. 2019 379Sánchez et al. Pregnancy establishment in cattle.
Figure 1. Use of an ex-vivo uterine endometrial explant-conceptus co-culture system to elucidate conceptus-induced
effects on the endometrium both dependent and independent of interferon-tau (IFNT). Uterine explants taken from
the same uterus were exposed to (i) medium alone (control), (ii) 100 ng/ml recombinant ovine IFNT, (iii) a long Day
15 conceptus, or (iv) a short Day 15 conceptus. Numbers of differentially expressed genes indicated for each group
are relative to the Control. Modified from Sánchez et al. (2019b).
Differential response of endometrium ipsilateral and a separate experiment within the same study, ten Day 7
contralateral to the CL in vitro produced blastocysts were transferred into the
uterine horn ipsilateral or contralateral to the CL or into
Embryo transfer studies established that the both horns (i.e., bilateral) of synchronized recipient
incidence of embryo loss is higher following transfer to heifers. Reproductive tracts were recovered at slaughter
the uterine horn contralateral to the ovary containing the on Day 14 and the number and dimensions of recovered
CL compared to transfer to the ipsilateral horn (Christie conceptuses were recorded for each horn. Site of
et al., 1979). Whether these differences are manifest in embryo transfer did not affect recovery rate (48.0%,
conceptus growth and elongation in the critical window 168/350) or length of conceptuses. Thus, although
preceding maternal recognition of pregnancy is differences in gene expression exist between the
unknown. Knowledge of differences in gene expression endometrium of uterine horns ipsilateral and
between the uterine horns during the estrous cycle could contralateral to the CL in cattle, these differences were
further enhance our understanding of uterine receptivity not associated with a reduced ability of the uterus to
and the process of conceptus elongation, key events for support conceptus survival or development to Day 14
the maternal recognition of pregnancy and, in turn, after embryo transfer on Day 7.
successful pregnancy establishment. In a follow-on study, we asked whether the
We hypothesized that differences in the endometrium from the uterine horn ipsilateral or
endometrial transcriptome between the ipsilateral and contralateral to the CL responds differently to an
contralateral horns throughout the cycle exist, and those elongating conceptus. Bagés-Arnal et al. (2019)
differences would be correlated with differences in compared the local response of the ipsilateral and
conceptus elongation after embryo transfer (Sánchez et contralateral endometrium to a Day 14 conceptus.
al., 2019a). Endometrial samples from both horns were Although no differences in gene expression were detected
collected from synchronized heifers slaughtered on Day between ipsilateral and contralateral endometrium, the
5, 7, 13 or 16 post-estrus and subjected to RNA response of the endometrium to a Day 14 conceptus was
sequencing. Main findings were that: (i) day of the distinct in each uterine horn. Interestingly, more genes
estrous cycle contributed to the greatest variation in the were differentially expressed in the contralateral than in
endometrial transcriptome; (ii) there were many more the ipsilateral endometrium after exposure to a conceptus
altered genes between the uterine horns ipsilateral and 239 vs. 61 DEG, respectively). Many of the biological
contralateral to the CL in the early (Day 5 and 7) as processes enriched in the DEG between both horns in
compared to late (Day 13 and 16) luteal phase; (iii) response to a conceptus were associated with immune
signalling pathways regulating pluripotency of stem response and response to stimuli. This observation is
cells were highly dysregulated when both uterine horns consistent with the study of Moraes et al. (2018), where
were compared, regardless of the day of luteal phase. In relatively few differences were detected in the
380 Anim. Reprod., v.16, n.3, p.376-385, Jul./Sept. 2019Sánchez et al. Pregnancy establishment in cattle.
endometrial transcriptome of non-pregnant high-fertile, (Simintiras et al., 2019a) - the greatest differences
subfertile and infertile heifers; however, the response of observed throughout the study - suggestive of a key role
the endometrium from high-fertile and subfertile for these metabolites in sustaining, in addition to
animals to pregnancy was remarkably different (3422 initiating, conceptus elongation.
vs. 1095 DEG, respectively). Secondly, sub-pathway enrichment and
These data extend those of Sánchez et al. representation analyses revealed that metabolic cascades
(2019a) describing temporal changes in the of likely importance to conceptus elongation-initiation
transcriptome of the endometrium ipsilateral and revolve around phospholipids, polyamines, and purines.
contralateral to the CL during a nonpregnant estrous Regarding the former, membrane biogenesis is
cycle by describing differential response of the intuitively essential to the ~30-fold increase in
endometrium in both uterine horns to an elongating trophoblast length between Days 12-15 (Betteridge et
conceptus. The large difference in the number of DEG al., 1980; Brooks et al., 2014). As 47% of identified
between the endometrium ipsilateral and contralateral to lipids were intricately linked to membrane biogenesis, it
the CL in response to a Day 14 conceptus may be seems reasonable to suspect that endometrial lipid
related to the differences in P4 concentrations during the secretions contribute to conceptus membrane fusion,
first days after ovulation (Takahashi et al., 2016), since, and, thus, elongating conceptus membrane biogenesis is
as mentioned earlier, P4 is one the major regulators of not entirely de novo (Simintiras et al., 2019b). The
the uterine receptivity through changes in the latter, polyamines and purines, are discussed below
endometrium transcriptome. within the context of adenosine monophosphate
signaling.
Uterine lumen fluid compositon Thirdly, P4 supplementation amplified the total
mean metabolite abundance on Day 14 (P ≤ 0.0001);
The composition of ULF during the however, just 19 metabolites (8.2% of total) were
preimplantation period has been extensively studied in elevated (P ≤ 0.05) on Day 14 in high vs. normal P4
sheep (see review by Bazer et al., 2015 and references heifers, and are, therefore, largely responsible for
therein). Data in cattle are more limited although raising the mean (Simintiras et al., 2019d). The
various studies have reported on aspects of ULF biochemical properties of the these ‘selectively’
composition under various physiological states (Mullen amplified 19 metabolites - including glucose (primary
et al., 2012; Faulkner et al., 2013; Forde et al., 2015). fuel source), trimethylamine-N-oxide (protein-
We recently metabolically interrogated ULF stabilizing osmolyte), and phenol sulfate (relatively
flushes on Days 12-14 - the window of conceptus metabolically inert yet acidic molecule) - (i) support the
elongation-initiation - from cyclic heifers, either (i) notion that optimal conceptus elongation is contingent
supplemented with P4 on Day 3 post-estrous (high P4 on biophysical and physicochemical, in addition to
cohort), or (ii) not (normal P4 cohort; physiological metabolic, cues, and (ii) contribute to the generation of
control). The former group is an established model of our hypothesis pertinent to the molecular bases of
conceptus elongation rate acceleration (Carter et al., conceptus elongation initiation, discussed below.
2008; Clemente et al., 2009; O’Hara et al., 2014). These findings combined, coupled with
Given that conceptus elongation coincides with a period previous data on the enzymatic profile of bovine ULF
of significant bovine pregnancy loss, our aim was to (Muñoz et al., 2012; Forde et al., 2014), give rise to the
achieve a better understanding of the biochemical hypothesis that conceptus elongation internally hinges
landscape surrounding the peri-elongation conceptus. on 5' adenosine monophosphate-activated protein kinase
Over 5000 metabolites were screened for by high- (AMPK) and peroxisome proliferator-activated receptor
throughput untargeted ultra-high-performance liquid gamma (PPARγ) activity, and is modulated by glucose,
chromatography tandem mass spectroscopy, with 233 adenine, and adenosine mono- (AMP), di- (ADP), and
consistently identified, clustering within 8 super- tri-phosphate (AMP) influx (discussed in Simintiras et
pathways: amino acids, carbohydrates (Simintiras et al., al., 2019d). Additional observations worth highlighting
2019a), lipids (Simintiras et al., 2019b), cofactors, include: (i) that total ULF metabolite abundance (Fig.
vitamins, nucleotides, peptides, energy substrates, and 2A) is not indicative of activity in terms of total day
xenobiotics (Simintiras et al., 2019c). A global analysis effects (Fig. 2B), P4 effects (Fig. 2C), or day by P4
of this dataset revealed three core ‘strategies’ likely interactions (Fig. 2D), and (ii) the identification of a
utilised by the bovine endometrium to facilitate plethora of microbiome-associated molecules in ULF,
conceptus elongation, discussed below. some of which were responsive to P4 (Simintiras et al.,
Firstly, indicative of the changing biochemical 2019c), highlights a need for further research into the
requirements of the conceptus around the initiation of influence of the uterine microbiome in uterine
elongation, a metabolic shift in the ULF of normal P4 metabolism and maternal-embryo communication.
heifers after Day 12 was observed (Simintiras et al.,
2019d), to which fructose and mannitol/sorbitol were Conclusion
central. More specifically, only these two metabolites
increased on Days 13 and 14 vs. 12 within the normal The period of early embryo development and
P4 group. Moreover, fructose and mannitol/sorbitol pregnancy establishment is fascinating. This complex
were elevated by 18.4 and 28.4-fold, respectively, in the process encompasses ovulation, fertilization, blastocyst
ULF of high vs. normal P4 heifers on Day 12 formation and growth into an elongated conceptus,
Anim. Reprod., v.16, n.3, p.376-385, Jul./Sept. 2019 381Sánchez et al. Pregnancy establishment in cattle.
pregnancy recognition signalling, and development of exhibited amongst conceptuses which is independent of
the embryo and placenta. Despite the aforementioned the uterus and may point to variation in oocyte and early
advances in the field, there is still much to learn. The embryo quality. Furthermore, the role of the sire in
precise drivers of conceptus elongation remain determining embryo quality and in conceptus
unknown. While the process is dependent on the uterus development is only beginning to be appreciated
- it does not occur in vitro - there is significant variation (Ortega et al., 2018).
Figure 2. Breakdown of metabolites identified in uterine luminal fluid, by super-pathway, on Days 12-14 of cyclic
heifers in terms of total: (A) abundance, (B) day effects, (C) progesterone (P4) effects, and (D) day by P4
interactions. Adapted from Simintiras et al. (2019d).
periovulatory plasma progesterone concentration in the
Author contributions ewe. J Reprod Fertil, 87:23-32.
Barnwell CV, Farin PW, Ashwell CM, Farmer WT,
All authors (JMS, CAS, PL) contributed to the Galphin SP, Farin CE. 2016. Differences in mRNA
writing and revising of the manuscript. populations of short and long bovine conceptuses on
Day 15 of gestation. Mol Reprod Dev, 83:424-441.
Conflict of interest Bartol FF, Roberts RM, Bazer FW, Lewis GS,
Godkin JD, Thatcher WW. 1985. Characterization of
Authors declare no conflicts of interest. proteins produced in vitro by periattachment bovine
conceptuses. Biol Reprod, 32:681-693.
Acknowledgments and Funding disclosure statement Bauersachs S, Ulbrich SE, Zakhartchenko V, Minten
M, Reichenbach M, Reichenbach H-D, Blum H,
This work was supported by grants from Spencer TE, Wolf E. 2009. The endometrium responds
Science Foundation Ireland (to PL; Grant number: differently to cloned versus fertilized embryos. Proc
13/IA/1983), an Irish Research Council Government of Natl Acad Sci USA, 106:5681-5686.
Ireland Postdoctoral Fellowship (to CAS; grant number Bauersachs S, Ulbrich SE, Reichenbach HD,
GIOPD/2017/942), and a University College Dublin Reichenbach M, Büttner M, Meyer HH, Spencer TE,
Career Development Award (to CAS; Grant number: Minten M, Sax G, Winter G, Wolf E. 2012.
CDA54580). Comparison of the effects of early pregnancy with
human interferon, alpha 2 (IFNA2), on gene expression
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